The present invention relates to the fields of electrophysiology, molecular biology and molecular medicine, and more specifically to regulation of neuronal membrane potentials and excitabilities involved in BD and ADHD by polypeptides in the diacylglycerol signaling pathway, and modulation of the same.
The present invention relates to a method of identifying the modulators of the membrane potentials of patients' cells that could serve as the drug targets for decreasing the membrane potential ratio (MPR™) values in ADHD patients and for lowering the MPR™ values to the levels of the MPR™ values of negatives. The invention relates to a method of identifying the modulators of the membrane potentials of patients' cells that could serve as the drug targets for increasing the MPR™ values in BD patients and for raising the MPR™ values to the levels of the MPR™ values of negatives. This invention further identifies the DAG signaling pathway as a principal signaling mechanism that modulates the MPR™ values. Furthermore this invention identifies modulators such as compounds and polypeptides along this pathway as diagnostic markers and drug targets for BD and ADHD. They include DAG and its associated enzymes and kinases, PKC isoforms and associated enzymes and kinases, and Ca+/CaM and its associated enzymes and kinases.
It is well recognized that mental disorders are caused by the malfunction of the neurons in the brain. Neurons communicate with each other through electrophysiological signals. These signals are generated and modulated by the membrane potential and the excitability of the neurons. The identification of the molecules that modulate the signaling pathways in the neuronal cell is essential in diagnosing and treating mental illness. The membrane potential is the electrical potential difference (voltage) across a cell's membrane. Membrane potential results from the action of K+ ion channels present in the membrane which along with the Na, K-ATPase enzyme maintain viable ion concentrations inside the cell.
Unlike most cells, neurons are electrically active and use changes in membrane potential for fast communication with other neurons. Neurons process and transmit information in the form of electrical signals. K+ ion channels in the neuronal membrane set the membrane potentials and the excitability. These signals are then processed, amplified and transmitted to the synapse releasing the neurotransmitters. These transmitters again send a signal through their specific G-protein coupled receptors (GPCR) in the membrane of the target neuron. The GPCRs transmit these signals through two primary signal transduction pathways that process and transmit this signal to the K+ ion channels in its membrane. These two pathways are the cAMP signaling pathway and the DAG signaling pathway (Nahorski S. R. British Journal of Pharmacology (2006) 147, S38-S45).
Calculations of the membrane potentials (MP) using Goldman-Hodgkin-Katz equation showed that lithium would depolarize the membrane potentials (Thiruvengadam, A. Journal of Affective Disorders 65 (2001) 95-99). This result led to the hypothesis that lithium's therapeutic efficacy was due to this depolarizing effect. This result was supported by earlier experimental and clinical results (Yonemura, K, and Sato, M, The Japanese Journal of Physiology, 1967; 17: 678-97), Grafe, et al, Brain Research, 1983; 279: 65-76 and El-Mallakh, et al, J. Affective Disorders, 1996; 41: 33-3). Thiruvengadam (Focus on Bipolar Disorder Research ISBN 1-59454-059-4 Editor: Malcomb R. Brown, pp. 15-35 © 2005 Nova Science Publishers, Inc.) further showed that lithium not only depolarizes the membrane potential but also reduces the excitabilities of neurons. Measurement of membrane potentials of cultured lymphoblasts collected from BD patients showed that the membrane potential was hyperpolarized confirming the measurements of El Mallakh et al. In order to use the membrane potential as a diagnostic marker for BD, a ratiometric method was developed and used successfully for diagnosing BD patients (U.S. Pat. No. 7,425,410 B2 which is incorporated herein in its entirety) using their red blood cells (RBC). This method involves the measurement of MP from changes that occur in the Na+K+ ATPase activity in cells, in two buffers and taking the ratio of these two MPs. These experiments involve a test buffer that contains no K+ ions but contains ethyl alcohol (EtOH). The membrane potentials are measured in the test buffer and compared with the membrane potentials measured in a reference buffer without EtOH. This ratio is called the membrane potential ratio (MPR™). It was further discovered that the membrane potential ratio (MPR™) could also be used to diagnose ADHD patients (U.S. Pat. No. 7,906,300 B2 which is incorporated herein in its entirety). To date, more than 550 patients have been tested using membrane potential ratio (MPR™). A summary of these test results is shown in FIG. 1. The membrane potential ratio (MPR™) values for BD patients were significantly lower than that for negatives (including normals, unipolar depressives, and schizophrenics). On the other hand, the membrane potential ratio (MPR™) values for ADHD patients were significantly higher than that for negatives as shown in FIG. 1.
It is essential to understand the biological basis for these differences in order to determine the scientific mechanism(s) and pathway(s) responsible for the differences in the membrane potential ratio (MPR™) among the three groups and to elucidate the pathophysiology of these illnesses.
Currently drug development efforts utilize neurotransmitters, their release, uptake and activation of their receptors as therapeutic drug targets for BD and ADHD. However, these drug targets do not serve as diagnostic biomarkers. The mechanism of action of drugs like lithium, amphetamine and anticonvulsants such as valproate and carbamazepine are not well understood. The results that the membrane potential ratio (MPR™) values for BD patients are significantly lower than those for negatives (including normals, unipolar depressives, and schizophrenics) and that the membrane potential ratio (MPR™) values for ADHD patients are significantly higher than those for negatives (as shown in FIG. 1) are very significant since they for the first time directly connect the specific patients with mental disorders to the specific biomarkers via changes that occur in the Na+K+ ATPase regulation and membrane potential changes therein. An understanding of the signaling pathway and the identification of the polypeptides and compounds modulating Na+K+ ATPase regulation was essential in advancing and enabling the diagnosis of BD and ADHD. To date, there are no studies in the art that connect the signaling pathway, the polypeptides involved, electrophysiological parameters such as membrane potentials and excitabilities with mental disorders such as BD and ADHD. The present invention accomplishes this objective leading to better diagnostics and therapeutics.
CAK Channels and Membrane Potentials In RBC: Although the expression of one of the small conductance family of CAK channels in RBC has been known since 2003 (Hoffman et al, PNAS, 100(12): 7366-7371 (2003); which is incorporated herein in its entirety), there has been no measurement of the membrane potential in RBC much more an observation of the differences among three groups of patient populations (negatives, BD and ADHD). The observation that EtOH hyperpolarizes the membrane potentials has been recognized in the art as a new discovery. However, only experiments using channel blockers, quinine and clotrimazole in RBC established this fact. No other literature in the art shows a connection between calcium-activated potassium (CAK) channels and ethanol, nor the involvement of CAK channels and cellular membrane potentials.
Ca2+/CaM Activation of CAK Channels, EtOH and Membrane Potentials in RBC: It is well-known in the art that CAK channels are activated by calcium-calmodulin (Ca2+/CaM). However, there is nothing in the art demonstrating the modulation of membrane potentials by either ethanol or by a calmodulin (CaM) activator such as calmodulin (CaM) Kinase II.
PKC, CaM and membrane potentials: It is well-known in the art that PKC through the DAG signaling pathway activates calmodulin (CaM). However, no studies in the art have shown that DAG signaling pathway modulates the CaK channels and membrane potentials.
DAG, CAK Channels and MP: DAG and the DAG signaling pathway has not been shown or understood in the art to have an effect on membrane potentials, much more be involved in the diagnosis of BD and ADHD. For instance, U.S. Pat. No. 6,593,121 2003 to Caricasole et al. (which is incorporated herein by reference in its entirety) relates to human diacylglycerol kinase proteins (hDAGK), and does not address membrane potential ratio (MPR™) differences nor modulation of the membrane potential ratio by the DAG pathway. Baum et al., Mol. Psychiatry 13(2): 197-207 (2008) was a genome-wide association study that implicated the diacylglycerol kinase eta (DGKH) and several other genes in the etiology of BD. However, Baum did not recognize that membrane potential ratios (MPR™) may be modulated by the DAG signaling pathway.
Thus, the present investigator identified an unaddressed need in the art, to determine the signaling pathway(s) and drug targets of the signaling pathway(s) that regulate cellular membrane potentials to provide a more specific means of identifying compounds that bind to these drug targets and modulate the interaction of these drug targets. These signaling pathways and drug targets can then be used for diagnostic and therapeutic purposes. For instance, the drug targets may be polypeptides involved in the diacylglycerol signaling pathway.
The present invention satisfies this need and provides related advantages as well. For example, this invention traces the pathway for BD and ADHD from the G-protein coupled receptors (GPCR) to the K+ channel in patients' cells. The present discovery provides a better understanding of the pathophysiology of these disorders and a better means to diagnosis and treat BD and ADHD.